Occult hepatitis B infection is defined as the presence of HBV DNA in serum and/or the liver tissue without detectable HBsAg with or without anti-HBc or anti-HBs antibodies. In Egypt many studies on occult hepatitis B have been conducted and this infection is well established in various patients groups. This short review sheds light on the occurrence of occult hepatitis B infection among different disease states in Egypt. The coexistence of occult hepatitis B with hepatitis C infection is of particular importance because of its added co-morbidity of liver enzymes elevation, increased severity of liver disease and increased risk of hepatocellular carcinoma. Patients on regular hemodialysis and those exposed to blood transfusion are at high risk of acquiring this form of infection. The highest prevalence of occult hepatitis B virus in Egypt was reported among patients with hepatocellular carcinoma and similar to the scenario for classic hepatitis B infection, genotype D is the most prevalent genotype.

Hepatitis B virus (HBV) infection is a global health problem. Itsd patterns of transmission vary greatlythroughout the world.[1] Furthermore, the consequences of chronic HBV infection represent a major burden for health care systems because a large proportion of these patients go on todevelop cirrhosis and hepatocellular carcinoma (HCC).[2] The prevalence of HBsAg in Egypt is of intermediate endemicity(2–8%).[3] Nearly 2-3 million Egyptians are chronic carriers of HBV. In Egypt HBV transmission is apparently a mixture of horizontal and perinatal transmission. However, the majorityof HBV infection is acquired by the former route.[2]

Occult hepatitis B virus infection (OBI) is one of the mostchallenging topics in the field of viral hepatitis with its virological and clinical relevance being debated for more than30 years.[4] Occult hepatitis B virus infection is defined as the presence of HBV DNA in serum and/or liver tissue withoutdetectable HBsAg with or without anti-HBc or anti-HBs antibodies outside the pre-seroconversion window period.[5]

Prevalence of OBI varies in different studies due todifferences in: (1) method of detection, including PCR primer selection and sensitivity of the assay; (2) patient recruitment,for example blood donors with/without anti-HBc; (3) patients from countries highly endemic for HBV are more likely todevelop OBI; (4) prevalence may vary according to the nature of the biological material tested, with a higher proportion for liver compared to serum specimens and also the amount ofDNA in the tested sample may influence prevalence rates of OBI;[6] and (5) a spurious increase of OBI prevalence rates may also noticed when kits with low sensitivity for detection ofHBsAg are used. Hence to correctly test for HBsAg,international standard samples with known quantities of HBsAg are required.[7]

Since the diagnosis of OBI is dependent on detection ofHBV DNA by PCR, which is an expensive tool and the Egyptian budget for health care and scientific research is very low, manyEgyptian studies depend on anti-HBc positivity for diagnosis of OBI leading to missing of HBV DNA positive/anti-HBcnegative cases and consequently underestimating the problem. Although there are no accurate estimates about the prevalence of OBI in different population groups in Egypt, this review isan attempt to shed light on available studies examining OBI prevalence and its impact on different population groups inEgypt (Table 1).

OBI and genetic mutations

Replication-defective mutants of HBV have been detected inthe circulation of symptom free individuals as early as 1987.[8] In HBV sequences obtained from serum samples of HBsAgseronegative carriers, a plethora of mutations has been observed. Point mutations, deletions and splicing alternativeshave been associated with OBI, but it is unclear weather these mutations are a cause or a consequence of this occult infection. Many of these OBI are associated with mutations in the S geneand/or regions governing the regulation of S gene expression, but they have also been documented in the core (C) andpolymerase (P) genes.[9] Naturally occurring pre-S deletion mutants were shown to influence not only the liver disease progression but also chronic hepatitis B therapy. Mutationsthat change the antigenicity of HBsAg or decreases its synthesis may be the cause of lack detection of HBsAg inpatients with OBI.[10]

It was shown that OBI individuals are infected by viralvariants either producing an antigenically modified HBV S protein undetectable by the available HBsAg assays,[11] orcarrying mutations capable of inhibiting S gene expression and/or viral replication.[12] Pre-S variants and deletions werealso found in occult isolates. One study showed that deleted regions were found more frequently in the 3' terminus of pre-S1which contains several immune epitopes and functional domains such as the site for dual topology, a nucleocapsid binding site, and the S-promote.[13] Removing sites for dualtopology and encapsidation would lead to incorrectconformation of the large HBsAg and decrease secretion ofHBsAg and viral particles.[14] Another important consequence of the deletion is that removing the S promoter alters smallHBsAg expression and leads to HBsAg seronegativity.[15] Chaudhuri et al[12] found a similar pre-S variant from occult HBV carriers and demonstrated that deletion of the S promoter alters the ratio of large and small surface proteins and decreases the circulating HBsAg level. Therefore, the existence of HBsAgnegative HBV infection may result from deletion of the Spromoter in the pre-S region, thus decreasing the quantity of HBsAg secretion below the sensitivity of a standard test butjust enough for viral assembly.[10]

Co-infection with hepatitis C virus (HCV)

In Egypt the rate of anti-HCV positivity is 15% (although rangesfrom 6-28% are proposed) and 5% of community is positive for both anti-HCV and HBsAg and/or anti-HBc, although the realestimated prevalence of HBV/HCV dual infection is unclear.[16] DNA positive OBI was evaluated in Egyptian chronic HCV patients on pegylated interferon/ribavirin therapy and wasnoted to occur at a rate of 3.9%,[17] this rate is increased to 10% in the subgroup of patients who developed elevated liverenzymes while on therapy,[18] and the rate increased to 20% in the subgroup of patients who had elevated liver enzymeswithout therapy.[19] The prevalence of OBI in chronic HCVpatients was higher in subjects having either anti-HBs or anti- HBc or both.[20,21] Serological findings in patients with OBI andHCV co-infection revealed that 35% of people were anti-HBs positive, 42% were anti-HBc IgG positive and 22% werenegative for both.[22]

In one study from Egypt, the prevalence of OBI in chronicHCV was projected to be 50% depending on anti-HBc positivity only, although only 60% of these tested positive for HBV DNAby PCR.[23] This is not in agreement with the consensus of HBV DNA positivity as a prerequisite for diagnosis of OBI.[24] Inanother study on Egyptian chronic HCV patients, serum HBV DNA was detected in 22.5% of anti-HBc positive chronic HCV patients and nothing was detected in anti-HBc-negative chronicHCV patients.21 In another study OBI was detected in 19 patients with HCV infection (out of 40), it was higher in patientspositive for both anti-HBs and anti-HBc (50%) than those having anti-HBc alone (20%).[24] Although this was not the observed trend in other studies.[18,25] Emara et al[18] examined 155chronic HCV patients on pegylated interferon/ribavirin combination and found six patients positive for HBV DNA.

OBI could not be predicted by serological markers of HBVinfection because only two out of the six patients with detectable HBV DNA had anti-HBc antibodies and none had anti-HBsantibodies.[18] While one study did not notice and variation in the prevalence of occult HBV infection with age or sex,24 anotherstudy found OBI prevalence higher in younger chronic HCVpatients.[18]

OBI/HCV viral interference

Preliminary data suggest that HCV core protein might inhibitthe replication of HBV.26 A similar effect was also attributed to NS2 protein of HCV.[27] Hepatitis C infection reduces theexpression of HBV proteins in the liver.[28] Conversely, there is some evidence that HBV replication might interfere with HCVviral load[29] and even lead to a higher chance of HCV RNAclearance.[30] Both events have been demonstrated in the study of Rodriguez-Inigo et al[31] where they proved that both HCVand HBV can coexist in the same hepatocyte. Furthermore, HBV DNA levels in co-infected hepatocytes were lower thanthose in cells infected only with HBV. This finding supportsthe hypothesis of inhibition of HBV replication by HCV. Also, HCV RNA levels were lower in co-infected cells than in cellsinfected with HCV only, suggesting that HBV may also inhibit HCV replication. However, it seems that inhibition of HBV replication by HCV is more common than the opposite.

Co-infection by both occult hepatitis B and occult hepatitisC does occur and contrary to the fact that dual chronic HBV and HCV leads to a more severe disease than mono-infection[32]dual occult infection was not associated with more severe liver disease in comparison to single occult mono-infection. Thisdata suggests that in occult infections both viruses tend to inhibit each other reciprocally. This can be explained by: (1) in chronic dual infection, a more vigorous immune responseagainst the infected hepatocytes is the cause of severe liver damage, (2) in occult infection antigen expression on infectedhepatocytes may be too low to trigger sufficient cytopathic antiviral immune response so as to induce a greater liver damage in occult dual infection compared to a single occult infectionand dual chronic infections.[33]

Impact of OBI on HCV disease states

Flares of liver enzymes

Several investigators have studied the association betweenOBI and increased liver enzymes in serial serum samples. Zignego et al[34] found that ALT relapses following interferontreatment for chronic HCV were associated with HBV viremia in some cases. Fujiwara et al[35] found that HBV viremia in chronicHCV patients with OBI did not correlate with liver enzyme flares. Kannangai et al[36] demonstrated that episodes of viremia in OBI/HCV dually infected non-treated intravenous drug userscan be associated with transient but significant flares in liver transaminases. This notion supports the hypothesis that OBIreplication may be a cause of hepatocyte injury, although this is contrary to the trend in literature about the lack of strong correlation between ALT/AST levels and OBI in cross sectionalstudies.[37,38]

The ALT levels are usually mildly raised in OBI and HCVco-infection[20,39] and seem to be either equivalent[40] or mildly raised[19,20,41] than in HCV mono-infection. The effect of OBI onliver enzyme flares has not been studied extensively in the Egyptian literature. One study found that OBI is not a cause ofliver enzyme flare during pegylated interferon/ribavirin therapyfor chronic HCV.[19] Another recent Egyptian study compared the frequency of OBI in chronic HCV with normal or slightlyraised liver enzymes with another group of patients with liver enzyme flare (>5 folds) and found OBI in 63.3% of patients with enzymes flare in comparison to 13.3% in the normalenzymes group.[25]

Increased severity of liver disease

In a surprising contrast to the mild inflammation noticed inOBI/HCV co-infection[42] most studies have demonstrated an increased prevalence of advanced fibrosis/cirrhosis in thesecases.[41-43] Such occult HBV co-infection is associated with more severe liver disease and higher HCV viral load.[44] In one studyfrom Egypt the presence of OBI was found to aggravate the severity and deterioration of liver disease in patients with chronic HCV.[45] In contrast other studies found no associationbetween OBI and the degree of liver necroinflammation and fibrosis.[37,46] In agreement with these latter findings is our studywhere we found less hepatic fibrosis in OBI/HCV co-infected patients in comparison to HCV mono-infections.

Necroinflmmatory activity also showed no significantdifference, although these results may be due to the small number of patients in the co-infection group of our study.[17]Altered response to antiviral treatment Some studies suggest a negative influence of OBI on theresponse to standard interferon therapy in chronic HCV infection irrespective of HCV genotypes[20,43,44,47,48] but thisobservation needs to be confirmed in HCV populations treated with the current gold standard, pegylated-interferon/ribavirin combination therapy.[4] Consequently we conducted a crosssectional study to evaluate the prevalence and impact of OBI in HCV patients on antiviral therapy. Out of the HCV patientsunder treatment, six were HBV DNA positive (OBI) and therewas no statistically significant difference in response to HCV treatment with pegylated interferon/ribavirin combinationtherapy.[17]

Increased risk of HCC

There is an elevated risk of HCC in HCV patients because thissilent infection can affect the progression of liver disease towards HCC development.[49] Studies have found highproportion of HCV related HCC patients suffering from concomitant OBI.[50]Anti-HBc antibody and HCVAdvanced hepatic fibrosis has been reported in patients with isolated anti-HBc and chronic HCV infection.[47] Anti-HBc antibody was linked to more severe liver disease and poorresponse rates to interferon therapy for HCV in international[29] and Egyptian studies alike.[18,21,51]

Occult hepatitis B and blood transfusion

The first recorded evidence of OBI and possible transmissionoccurred via blood transfusion.[52] This was further confirmed in the chimpanzee model when cloned full-length HBV-DNAfrom patients with OBI was confirmed to be infectious and generated a typical HBV infection.[53] Thus HBsAg non-reactiveblood donations containing HBV DNA have to be considered infectious.[54] In Egypt prevalence of anti-HBc antibody in HBsAg negative blood donors was found to be 13.3% of which10% were HBV DNA positive.[55] In another large Egyptian study among the accepted blood units for donation in blood banks,anti-HBc antibody was found in 78/712 units (10.96%). HBV DNA was detected in 9/78 (11.54%) of the anti-HBc-positive units and thus OBI was detected in 9/712 (1.26%) of theaccepted blood donations. Implementing anti-HBc test to the routine assay for the forthcoming two decades would certainlyeliminate the possibly HBV-infected units. Rejection of these units will be beneficial to decrease the risk of HBV transmission with its potential consequences particularly inimmunocompromised recipients.[56] The prevalence of OBI is much higher in patients exposed to multiple bloodtransfusions[57] especially when immunocompromised.[58] OBI was reported in 21% of Egyptian HCV infected children withhematological disorders and malignancies.[59]

Occult hepatitis B and hemodialysis

HBV and HCV share common routes of transmission and hencepatients on hemodialysis are at increased risk of exposure to infected blood and instruments and acquiring both infections.In one study from upper Egypt 4% of hemodialysis patients were found to have OBI. The prevalence did not significantlyvary between hemodialysis patients with or without HCV coinfection. Another study from lower Egypt reported lower prevalence of OBI in chronic hemodialysis patients and therewas no significant difference in prevalence between hemodialysis patients with (6.3%) or without (3.8%) HCV coinfection.

Risk of HBV reactivation

Any patient with OBI receiving systemic chemo- radio- orimmuno-therapy is potentially at risk of HBV reactivation,6 leading to the development of a typical hepatitis B infectionthat often has a severe and sometimes even fulminant clinical course.[61] The risk of reactivation is well documented in HBsAgpositive hemato-oncologic patients who require pre-emptiveantivir al therapy. All patients receiving chemo- and immunotherapy should be tested at least once for anti-HBc antibodiesbefore starting therapy and monitored periodically for ALT elevations. In case of ALT elevation, further diagnostic workup is required pending initiation of antiviral therapy uponestablishing the diagnosis of HBV reactivation.[62] In this context OBI patients under immunosuppression may show areactivation of viral replication when immunological reconstitution is achieved with a subsequent cytotoxic Tlymphocyte mediated hepatocyte injury.[63] Monitoring in such cases should be extended for months or even years after discontinuation of immunosuppression.[64] Patients prone to OBIreactivation are shown in Table 2.

OBI and organ transplantation

Transmission and reactivation of OBI is important in the settingof organ transplantation particularly with liver transplants. There is a risk of transmission of OBI through an orthotopicliver transplant from an OBI-seropositive (anti-HBc-positive) donor, in particular if the recipient is negative for all HBV serummarkers, because of the presence of viral strains in the hepatocytes which can be reactivated duringimmunosuppression.[65] Prophylaxis with antiviral agents such as lamivudine prevents hepatitis B of the recipient in most (but not all) of these cases.[66] Although theoretically possible, thereis no evidence of HBV transmission from occult HBVseronegative organ donors.[63] In immunocompetent recipientsthere is no evidence that anti-HBs containing components are infectious, even at low titers.[54] The risk of occult HBV transmission is very low after kidney, heart or bone marrowtransplant.[67] Although the risk of acquiring HBV infection after bone marrow transplantation is low, nevertheless, reactivationof the latent infection may sometimes occur and lead to fulminant hepatic failure (FHF). A case of FHF due to HBV was reported 22 months after bone marrow transplantation obtainedfrom patient found to be anti-HBs positive, HBsAg negative and only positive for intrahepatic HBV DNA prior totransplantation.[68]

Occult hepatitis B and hepatocellular carcinoma

HCC patients living in areas endemic for HBV were frequentlyfound positive for HBsAg and/or anti-HBc antibodies and this strong relationship was the first epidemiological evidence ofHBV-related oncogenic transformation.[69] Persistent HBV infection may have a critical role in development of HCC evenin HBsAg-negative patients.[24] Development of HCC in OBI patients seems to be related in most cases to the associated co-infection with HCV and to the presence of cirrhosis, althoughOBI mono-infection still bears an oncogenic potential.[24,50] The highest prevalence of OBI in Egypt was reported amongpatients with HCC.[24,50] A difference in OBI prevalence rates as per the biological matter tested, is reflected in an Egyptian study where intrahepatic occult HBV DNA was detected in62.5% cases, whereas serum occult HBV DNA was detected in only 22.5% of the same HCC patient group.[24]

OBI could accelerate the evolution to cirrhosis in HCVpatients,43 and cirrhosis is known to be the most important risk factor for the development of HCC.[37] The cryptic infectionappears to exert its pro-oncogenic role in HCV infected patients as well as in alcoholics and in individuals with cryptogenicliver disease.[64,70] In HCV-related HCC the prevalence of serum OBI was 47.4% (9/19). The prevalence of OBI was higher in patients with both anti-HBs and anti-HBc 2/4 (50%) antibodiesthan those with anti-HBc alone 1/5 (20%).[24] One of the markers expressed in HBsAg negative HCC cases is the expression of the HBV-X gene. Several studies[71-74] have reported the HBV-Xprotein in the malignant liver tissues. The X gene deregulates cell cycle control, interferes with cellular DNA repair andapoptosis and plays an important role in interaction with p53 and Rb gene.[71] One Egyptian study found that HBV DNA and HBV-X gene was positive in 24/42 (57.1%) cases of HCCpatients, candidates for liver resection, HBV-X gene expression was significantly higher in HCC patients than in the HCVinfected group.[64]

OBI and chronic liver disease

Occult HBV has been associated with more advanced fibrosis/cirrhosis.[21] About 72% of HCC patients with intrahepatic occult HBV had cirrhosis. Cirrhosis is considered an important riskfactor for the development of HCC.[20] Similarly it was found that HBV mixed genotype infections could probably be ofclinical significance in HBV-induced liver diseases. Prevalence of mixed genotype infections was found to be 15.7% especially in those with chronic active hepatitis.[49] One Egyptian studyobserved that anti-HBc positive/HBV-DNA negative patients showed a similar prevalence of severe liver disease to anti-HBc positive/HBV-DNA positive patients and a significantly higher prevalence than anti-HBc negative cases. This notion raises the clinical significance of isolated positive anti-HBcantibody in relation to liver disease,23 although in other studies no association was found between OBI and the degree of livernecroinflammation and fibrosis.[37,46]

Genotypes

HBV genotype D is the prevalent genotype among chronicHBV infection in Egypt.[2] In one Egyptian study on the role of OBI in HCC, intrahepatic HBV infections were attributedpredominantly to viral genotypes D and B that constituted 8/ 25 (32%) and 6/25 (24%) of cases, respectively. HBV genotypesA and C infections were the least observed and constituted 4% and 8% of the cases, respectively, compared to 100% of HBV genotype A and D mixed infections in the control group(HBsAg positive HCC patients) with no other genotypes observed. In addition, there was a relatively high prevalenceof mixed infections of 5/25 (20%) among the studied group. One case had both genotypes A and D, two cases had both Band D and two cases had genotypes B and C. No HBV genotype E or F was found in this study and furthermore,genotypes G and H were not determined.[24]

Acknowledgements

The author would like to thank Professor Amira Soliman forrevising this work.

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